ABSTRACT The success of molecularly targeted cancer therapy using tyrosine kinase inhibitors (TKIs) faces a number of difficult challenges. Foremost among these is the ability to identify subsets of different cancers that are uniquely sensitive to targeted agents, often identified by the presence of genetic markers implying [unreadable]dependence[unreadable] or `addiction[unreadable] to the targeted pathway. Equally important to the longterm success of these therapies is understanding and circumventing acquired drug resistance, which is a key limitation to their clinical effectiveness. Acquired resistance to drugs targeting growth factor receptors differs from resistance to genotoxic cancer chemotherapy, and may include both specific mutations in targeted receptors, as well as more complex functional alterations in signaling networks. Here we will use non-small cell lung cancer (NSCLC) cell line models that appear to faithfully recapitulate key signaling dependence of cancers with activating mutations in the Epidermal Growth Factor Receptor (EGFR) gene, identifying a subset of lung cancers with extreme sensitivity to EGFR TKIs. We outline three aims that address the acquisition of resistance in tumors that were previously sensitive to these agents: in Aim 1, we will generate cell line models for acquired resistance to [unreadable]second generation[unreadable] irreversible inhibitors of EGFR, and use genetic, signaling and functional analyses to dissect the underlying mechanisms. In Aim 2, we will use a high throughput shRNA screen of tyrosine kinases to identify candidate targets whose suppression may circumvent resistance to EGFR inhibitors. In Aim 3, we will use lentiviral knockdown/reconstitution experiments to quantitate oncogene dependence of drug resistant cells, both on the initiating EGFR mutation and on associated signaling pathways that contribute to acquired drug resistance. Together, these aims will provide important insight into critical mechanisms that underlie the acquisition of resistance to novel inhibitors targeting growth factor receptors in human cancer. Public Health relevance Understanding the mechanisms by which cancers that are sensitive to the new classes of targeted cancer therapies become resistant to these is critical to their eventual clinical success. Our approach is designed to dissect the molecular basis of resistance to these cancer drugs.